cover glass;, it can then be calculated about how many drops of the water mixture are to be added to the gelatine, in order to have a convenient number of cells in one drop of the gelatine mixture. This number is 20 to 30 cells if a Böttcher chamber with a ring of 30 mm. diameter is used. But after some time it will be possible to prepare the proper mixture without counting or calculation.

When a suitable mixture of yeast and sterile water has been thus prepared, several Böttcher chambers are sterilised in the flame and placed under a sterile bell jar or sterile beaker to protect them from dust; all these experiments are carried out in the sterile cupboard. A very small drop of sterile water is then placed on the bottom of each chamber and some vaseline melted in a small saucer over a flame. The edge of the ring of the Böttcher chamber is painted with the melted vaseline, the latter substance being used because, after solidifying, a completely homogeneous mass without air bubbles is obtained. The requisite cover glasses are then flamed and likewise placed under sterile bell jars or small beakers. Finally a flask with wort gelatine is placed on a water bath at 30 to 35° C. to liquefy the gelatine.

A little of the yeast water mixture is mixed with the proper quantity of liquefied wort gelatine in a globular flask, and after being shaken vigorously, the formation of air bubbles being avoided, a drop is taken out with a thin glass rod or a fine pipette and spread out in a thin layer on the cover glass. The latter is left under the sterile bell jar for some minutes until the gelatine has set. It is then placed with the gelatine layer downwards on one of the Böttcher chambers and pressed firmly round the edge so that the vaseline closes it completely. The edge is painted with a melted mixture of 2 parts of vaseline and 1 part of wax to prevent the cover glass slipping. The Böttcher

chambers are often set up, as mentioned previously, in such a way that the cover glass is fixed to the loose ring with fish glue, and the ring is then fastened to the glass slip by means of vaseline or with a mixture of wax and vaseline.

We proceed now to investigate the chambers with not too great a magnification. Some practice is required in finding the cells; and it must not be forgotten to investigate the gelatine layer through its whole thickness, so that any cells deeply embedded may not escape notice. When a wellisolated cell is found, its position is marked; this is done either by means of the object marker of Klönne and Müller, by the use of squared cover glasses, or by using a stage with a scale or fixed mark (see pages 28, 30 and 33).

When we have by some means marked as many cells as is desired, and have convinced ourselves that there are no other cells in their immediate neighbourhood, the chambers are put away at the temperature of the room or at 25° C. If precautions are not taken, water drops are usually formed on the under side of the cover glass, especially at the edge of the gelatine and, what is worse, on the gelatine itself. To prevent the formation of these water drops, the chambers are placed under a moist bell jar which has previously been brought to a temperature a little higher than that in which the chambers are to be kept. It is advisable to examine the chambers after twenty-four hours in order to confirm the isolated positions of the marked cells.

When the colonies have become large enough, they are cautiously transferred to the nutrient liquid (cf. page 97) either by means of a piece of platinum wire held by a pair of forceps or by a very thin glass rod, the point of which can be easily broken off in the liquid to be infected.

In order to be able to use the object marker of Klönne and Müller, already mentioned, the objective is unscrewed

Holm found

and the point of the object marker coated with a dye solution. Since the chambers are often placed under a moist bell jar as described, a colour must be chosen which can withstand moist air and does not spread. that a suitable colour may be prepared from 0-25 part of fuchsine dissolved in 20 parts of aniline and mixed with 20 parts of a xylol solution of Canada balsam. A drop of this colour is spread in a thin layer on a small glass plate such as a glass slip. The point of the object marker is now pressed against the medium, so that the edge of the opening is distinctly coloured. Care must be taken that the colour film does not spread over the opening; if this happens it can be easily removed by blowing through the other end of the apparatus. The object marker is now screwed on to the tube of the microscope, and so adjusted that the point almost touches the cover glass of the moist chamber. The tube is then screwed downwards very cautiously for a small distance by means of the micrometer screw so that the point of the object marker touches the cover glass, with which it is allowed to remain in contact for about ten seconds, after which it is raised again. A red ring is thus stamped on the cover glass, inside of which is the isolated cell under observation. It is not advisable to fit the object marker on to a revolving nose-piece, for it often happens that the field of view of the objective and the opening of the object marker do not exactly coincide, and the cell therefore lies outside the coloured ring.

Sometimes it is a little difficult to mark distinctly on the cover glass. This is caused partly by the point of the object marker not being quite plane and partly by the coloured liquid not having the right consistency. Under such circumstances it is found that the best way is to allow the first layer of colour placed on the point of the object marker to dry on and then to apply a new layer. The first dry layer

then acts as an elastic cushion. The use of this apparatus requires practice and, as may be inferred, a light hand to avoid breaking the cover glass.

Squared cover glasses, with or without numbers in the squares, may be used, as already mentioned, instead of the object marker. In the first case the squares can be made larger than in the latter. The chamber is set up as described above, the isolated cells being marked in the following manner: If there are numbers in the squares (see Fig. 6, page 30), the square with its number and the position of the cell with respect to this number is drawn on a piece of paper. A fixed point is thus obtained and it will be easy to find the cell again. If there are no numbers in the squares (Fig. 5, page 30), each square can be designated by means of two numbers, the one being the number of the horizontal row, the other the number of the vertical column which contains the square. For example 3, 4 means that square

which lies in the third horizontal row and in the fourth vertical column. In this case there must, of course, be only one cell in each square.

Lindner's Droplet Culture. The methods described above can, of course, be varied in several ways; one of these, for instance, is Lindner's droplet culture (1893). He diluted a wort culture until only one cell was found in every streak or dot which he made with a drawing pen on a cover glass. The cover glass was then turned over and fixed with vaseline on a hollow glass slip or on a Böttcher chamber and the preparation examined microscopically. Those droplets showing only one cell were marked with ink dots on the upper side of the cover glass. After a few days the growths have developed and those droplets which contain the growths originating in one cell are sucked up by means of a small piece of sterile filter paper; the latter is then placed on wort gelatine in a flask, and a drop of wort is added in order to

accelerate development. Instead of taking up the drop on filter paper, a little gelatine may be added; the whole is then taken up on a platinum wire or similar instrument. and introduced into the wort. Lindner accordingly begins with a liquid, then uses gelatine, and only after a growth has formed on the gelatine are the mass cultures prepared in flasks containing nutrient liquid.

Schönfeld's Method. Of the dilution methods that of Schönfeld remains to be described. According to him a dilution is prepared by means of liquefied culture gelatine, and small spots are placed on a cover glass from this gelatine mixture by means of a drawing pen. Each spot ought, as

When this method

far as possible, only to contain one cell. is further considered it will be seen that the spots ought only to be so large that the whole of each may be in the field of view when using a medium magnification, and it will be found necessary to add a little more gelatine so that the small gelatine spots may not dry up and in order that a growth may take place at all. The method is, as may be seen from the foregoing, a combination of those of Hansen and Lindner.

II. Physiological Methods.-It often happens that the organism which is to be cultivated in a pure state is present in comparatively small numbers, and the abovementioned dilution methods cannot then be applied. We must then resort to a physiological method. These methods, however, are by no means exact, and offer no certainty of obtaining a perfectly pure culture.

Fractionated Culture after Klebs and Others. A method in general use among the older physiologists and bacteriologists was a combination of an imperfect dilution method with a physiological method. Klebs's method, the so-called fractionated culture, is an example of this, as it consists in inoculating new sterile culture liquid with the